X-RAY CRYSTALLOGRAPHIC STUDIES OF TAQ RNA POLYMERASE

Rockefeller University, New York NY

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Abstract

Transcription is the major control point of gene expression and RNA polymerase (RNAP) is the central enzyme of transcription. Our long term goal is to understand the mechanism of transcription and its regulation. Determining three-dimensional structures of RNAP and its complexes with DNA, RNA, and regulatory, is an essential step. This is best accomplished with highly characterized prokaryotic RNAPs, especially because of the high degree of conservation of RNAP structure and function from bacteria to man. To this end, we recently purified, crystallized, and determined the 3.3 angstrom units-resolution X-ray crystal structure of a prokaryotic RNAP, core RNAP from the thermophilic eubacteria Thermus aquaticus (taq). This represents a major breakthrough in our work. Here we propose further structural studies, all aimed towards adding to our understanding of the enzyme's function and its regulation. Specifically, we propose to: 1. Complete the refinement of the taq core RNAP model 2. Determine the 3.0 angstrom units crystal structure of taq core RNAP complexed with antibiotic inhibitors. 3. Determine the structure of taq core RNAP complexed with antibiotic inhibitors. 4. Determine the structure of taq RNAP holoenzyme. 5. Determine the structure of a binary complex between taq core RNAP and template DNA. 6. Determine the structure of an active ternary elongation complex between taq core RNAP, DNA template, and RNA transcript. These studies will have a wide-ranging impact on studies of transciption.